Transcriptional regulation of high affinity IgE receptor γ-chain

ABSTRACT

Transcriptional regulatory regions and transcriptional regulatory factors for the human high affinity IgE receptor (FcεRI) γ-chain gene are specified and are targets for the development of transcriptional regulatory agents for the FcεRI γ-chain gene. The following are provided: DNA comprising the full length or a portion of the base sequence shown in SEQ ID NO:1, that regulates transcription of the human high affinity IgE receptor (FcεRI) γ-chain gene; and DNA comprising the full length or a portion of the base sequence shown in SEQ ID NO:2, that regulates transcription of the human high affinity IgE receptor (FcεRI) γ-chain gene. The present invention is promising for the development of novel agents for the prophylaxis/treatment of allergic diseases, autoimmune diseases, thrombosis, glomerulonephritis, and lupus nephritis.

FIELD OF THE INVENTION

The present invention relates to the regulation of the expression of thehigh affinity IgE receptor γ-chain and more particularly relates totranscriptional control of the high affinity IgE receptor γ-chain geneand to the utilization of this transcriptional control.

BACKGROUND

The high affinity IgE receptor (referred to below as FcεRI) expressed onthe cell membrane of mast cells and basophils is known to be a keyglycoprotein in the type I allergic reaction. When antigen-specificIgE's bonded to FcεRI are crosslinked by the corresponding polyvalentantigen (for example, cedar pollen antigen for individuals sufferingfrom cedar pollen allergy, dust mite antigen for individuals sufferingfrom dust mite allergies), the FcεRI's are clustered, the signaltransduction mechanism operates, and the mast cells undergo initialactivation. As a result, various chemical transmitters that evokeallergic inflammation, i.e., most prominently the histaminepreliminarily stored in cell granules, are released and the newsynthesis and release of leukotriene, prostaglandin, and so forth, whichare intracellular metabolites, is explosively induced, evoking a type Iallergic reaction.

In addition, cytokine secretion from mast cells is promoted by theclustering of FcεRI's on the mast cells, inducing the expression ofvarious adhesion molecules in the neighboring vascular endothelialcells. Eosinophils and lymphocytes in the blood aggregate by binding viathese adhesion molecules to the vascular endothelial cells at the siteof inflammation. The late allergic reaction is evoked as a result.Moreover, the FcεRI expressed by the Langerhans cells of the skin ispresumed to contribute to the pathogenesis of atopic dermatitis byantigen presentation and cytokine production.

Based on the preceding, a promising strategy for the development ofagents for allergy prophylaxis treatment is to target the FcεRI thatspecifically mediates type I allergy and thereby block signaltransduction from this receptor at the point of origin.

FcεRI is also known to participate in platelet activation andglomerulonephritis, and given this it is also promising to carry out thedevelopment of thrombosis and glomerulonephiritis by targeting FcεRI.

In humans, human FcεRI functions expressed on the cell surface as atetramer of an α-chain, a β-chain, and 2 γ-chains or as a trimer of anα-chain and 2 γ-chains. The extracellular region of the α-chainbinds-directly to IgE while the β-chain γ-chain participate in signaltransduction into the cell. The γ-chain assembles with the othermolecules, which have a ligand binding site, to form a receptor on thecell surface. When ligand binds to the receptor's ligand binding site,the γ-chain transduces the signal into the cell.

For example, the γ-chain has been reported to perform the function oftransmitting an activation signal into the cell in the FcεRI-mediatedinduction of allergic reactions (refer, for example, to Non-Patentdocuments 1 to 3). In addition, the γ-chain has been reported to inducethe platelet activation reaction by associating with the collagenreceptor GP VI on the platelet (refer, for example, to Non-Patentdocument 4).

The γ-chain is also a constituent element of the IgG receptors FcγRIIIand FcγRI and the IgA receptor FcαR, and the suggestion has also beenmade these FcR's participate in glomerulonephritis-(refer, for example,to Non-Patent document 5).

-   Non-Patent document 1 Ra C et al., Nature, 341:752-754 (1989);-   Non-Patent document 2 Blank U et al., Nature, 337; 187-189 (1989);-   Non-Patent document 3 Kinet J P, Annual Review of Immunology,    17:931-972 (1999);-   Non-Patent document 4 Konishi H et al., Circulation, 105(8):912-916    (2002);-   Non-Patent document 5 Suzuki Y et al., Kidney Int., 54(4):1166-1174    (1998)

However, on the subject of transcriptional regulatory regions for thehigh affinity human IgE receptor γ-chain, only the analysis by Brini A Tet al., in 1993 has been carried out, and to date no detailed analysisthat precisely identifies transcriptional regulatory elements and/ortranscriptional regulatory factors has been performed.

As a result of intensive investigations, the present inventors succeededin identifying, from within previously unanalyzed regions, regions thatparticipate in the transcriptional regulation of the human FcεRI γ-chaingene and in identifying transcription factors that bind to these regionsand were thereby able to achieve the present invention.

SUMMARY

That is, the present invention provides (1) a DNA comprising the fulllength or a portion of the base sequence shown in SEQ ID NO:1, thatregulates transcription of the human high affinity IgE receptor (FCεRI)γ-chain gene; (2) a method of regulating transcription of the FcεRIγ-chain gene, that regulates the binding of Sp1 with an FcεRI γ-chaingene transcriptional regulatory region comprising the full length or aportion of the base sequence shown in SEQ ID NO:1; (3) a method ofscreening for a compound, or salt thereof, that regulates the binding ofSp1 with an FcεRI γ-chain gene transcriptional regulatory regioncomprising the full length or a portion of the base sequence shown inSEQ ID NO:1; (4) a DNA comprising the full length or a portion of thebase sequence shown in SEQ ID NO:2, that regulates transcription of thehuman high affinity IgE receptor (FcεRI) γ-chain gene; (5) a method ofregulating transcription of the FcεRI γ-chain gene, that regulates thebinding of Elf-1 or GABP α/β heterodimer with an FcεRI γ-chain genetranscriptional regulatory region comprising the full length or aportion of the base sequence shown in SEQ ID NO:2; (6) a method ofscreening for a compound, or salt thereof, that regulates the binding ofElf-1 or GABP α/β heterodimer with an FcεRI γ-chain gene transcriptionalregulatory region comprising the full length or a portion of the basesequence shown in SEQ ID NO:2; (7) a DNA comprising the full length or aportion of the base sequence shown in SEQ ID NO:3, that regulatestranscription of the human high affinity IgE receptor (FcεRI) γ-chaingene; (8) a method of regulating transcription of the FcεRI γ-chaingene, that regulates the binding of C/EBP with an FcεRI γ-chain genetranscriptional regulatory region comprising the full length or aportion of the base sequence shown in SEQ ID NO:3; (9) a method ofscreening for a compound, or salt thereof, that regulates the binding ofC/EBP with an FcεRI γ-chain gene transcriptional regulatory regioncomprising the full length or a portion of the base sequence shown inSEQ ID NO:3. (10) a method of regulating transcription of the human highaffinity IgE receptor (FcεRI) γ-chain gene by controlling functionalregulation by at least one transcription factor selected from Sp1,Elf-1, GABP α/β heterodimer, and C/EBP or by controlling the interactionbetween or among transcription factors; (11) a vector that incorporatesthe full length or a portion of the base sequence shown in SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3; (12) a screening kit used in the screeningmethod according to the preceding (3), (6), or (9); (13) a drugcontaining a compound, or salt thereof, that regulates transcription ofthe FcεRI γ-chain gene and is obtained using the screening methodaccording to the preceding (3), (6), or (9) or the transcriptionalregulatory method according to the preceding (10); (14) the drugdescribed in the preceding (13), that is a prophylactic/therapeuticagent for allergic diseases or autoimmune diseases; (15) the drugdescribed in the preceding (13), that is a prophylactic/therapeuticagent for thrombosis; and (16) the drug described in the preceding (13),that is a prophylactic/therapeutic agent for glomerulonephritis or lupusnephritis.

Thus, the present inventors cloned the human γ-chain gene and using ahuman cell line carried out a reporter assay on the region upstream fromthe translation initiation point. The presence of two enhancer elementsin the nt-103/-75 region (here and in the following, the translationinitiation point is designated as base number +1) was elucidated as aresult, i.e., the region shown by SEQ ID NO:1, containing nt-98 to -96,and the region shown by SEQ ID NO:2, containing nt-84 to -82. Withregard to these elements, it was found by EMSA that Sp1 binds to theformer and that Elf-1 or GABP α/β heterodimer binds to the latter.

In addition, the region of SEQ ID NO:3, which contains nt-65 to -61 andexhibits homology with the C/EBP binding sequence, was found tocontribute to activation of the γ-chain promoter. On the occasion ofcarrying out reporter assays while inducing overexpression of variouscombinations of these transcription factors, it was shown thattranscription activation was synergistically increased and that the useof a plurality of the transcription factors resulted in cooperativeactivation of the γ-chain promoter.

Moreover, in view of the following facts as cited in the preceding: theγ-chain functions to transmit the activation signal into the cell duringinduction of the FcεRI-mediated allergic reaction, the γ-chain inducesthe platelet activation reaction by associating with the collagenreceptor GP VI on the platelet, and the γ-chain is a constituent elementof the IgG receptors FcγRIII and FcγRI and the IgA receptor FcαR andthese FcR's participate in the pathogenesis of glomerulonephritis, theexpression regulatory regions and control factors identified by thepresent inventors for the γ-chain gene are useful as targets for thedevelopment of prophylactic/therapeutic agents for, inter alia, allergicdiseases, autoimmune diseases, thrombosis, glomerulonephritis, and lupusnephritis. The present invention is also useful for genetic analysis inpersonalized medicine.

The genomic structure and base sequence of the human FcεRI γ-chain genehave already been elucidated (GenBank, accession number M33196, SEQ IDNO:4). The phrase, “comprising the full length or a portion of the basesequence shown in SEQ ID NO:1”, used in the present invention meanscomprising the main portion essential for transcriptional regulation ofthe FcεRI γ-chain gene and in particular comprising a sequence based onnt-98 to -96 (the translation initiation point is designated as basenumber +1).

The phrase, “comprising the full length or a portion of the basesequence shown in SEQ ID NO:2”, used in the present invention meanscomprising the main portion essential for transcriptional regulation ofthe FcεRI γ-chain gene and in particular comprising a sequence based onnt-84 to -82 (the translation initiation point is designated as basenumber +1).

The phrase, “comprising the full length or a portion of the basesequence shown in SEQ ID NO:3”, used in the present invention meanscomprising the main portion essential for transcriptional regulation ofthe FcεRI γ-chain gene and in particular comprising a sequence based onnt-65 to -61 (the translation initiation point is designated as basenumber +1).

The “high affinity IgE receptor γ-chain” referenced in the presentinvention was discovered as a high affinity IgE receptor γ-chain, butsubsequent findings have shown that, inter alia, other immunoglobulin Fcreceptors also contain it in common as a constituent element, and at thepresent time it is also known as “immunoglobulin receptor γ-chain”.

The present invention provides base sequences that participate intranscriptional regulation of the human FcεRI γ-chain gene andestablishes a method of screening for compounds and salts thereof thatinhibit γ-chain expression and also establishes a kit for use in thisscreening method.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the transcriptional regulatory activity according to areporter assay of the 5′ region of the human FcεRI γ-chain gene;

FIG. 2 shows the identification of binding factors by a gel shift assay,wherein (A) is a competitive test using unlabeled double-strandedoligoDNA (the added unlabeled double-stranded DNA was as follows: lanes3, 4; sequence identical to probe, lanes 5, 6; three bases substitutedin the probe sequence) and (B) is a test using antibody;

FIG. 3 shows the identification of binding factors by a gel shift assay,wherein (A) is a competitive test using unlabeled double-strandedoligoDNA (the added unlabeled double-stranded DNA was as follows: lanes3, 4: sequence identical to probe, lanes 5, 6: three bases substitutedin the probe sequence) and (B) is a test using antibody;

FIG. 4 shows the effect of site-directed mutagenesis on human FcεRIγ-chain gene promoter activity; and

FIG. 5 shows the effect of the overexpression of the differenttranscription factors on human FcεRI γ-chain gene promoter activity inHeLa cells.

DETAILED DESCRIPTION

Embodiments of the present invention will now be described. Thefollowing embodiments are examples for the purpose of explaining thepresent invention, but the present invention should not be construed aslimited only to these embodiments. The present invention can beimplemented using various modalities while preserving the essentialfeatures of the present invention.

The present invention, based on the determination of DNA regions thatparticipate in regulating transcription of the FcεRI γ-chain gene andthe identification of transcriptional regulatory factors that bind tothese regions, enables the construction of a method of screening forcompounds and salts thereof that inhibit expression of the FcεRI γ-chainand of a kit for use in this screening. The present invention therebycontributes to the development of agents for the prophylaxis ortreatment of allergic diseases, autoimmune diseases, thrombosis,glomerulonephritis, and lupus nephritis. For example, the development ofcompounds or salts thereof that inhibit γ-chain expression is possiblebased on the strategy of searching for substances that inhibit thebinding of the identified transcriptional regulatory factor Sp1 with thespecified gene region.

The aforementioned salt of a compound denotes, inter alia, a salt with aphysiologically acceptable acid (for example, an inorganic acid or anorganic acid) or a salt with a physiologically acceptable base (forexample, an alkali metal). Physiologically acceptable acid-adduct saltsare particularly preferred. Specific examples are salts withhydrochloric acid, phosphoric acid, hydrobromic acid, and sulfuric acidwithin the realm of inorganic acids and salts with acetic acid, formicacid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaricacid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid, benzenesulfonic acid, and so forth within therealm of organic acids.

An FcεRI γ-chain gene transcription-regulating compound or salt thereofthat is obtained using the screening method and screening kit accordingto the present invention is useful as an agent for theprophylaxis/treatment of allergic diseases, autoimmune diseases,thrombosis, glomerulonephritis, and lupus nephritis.

A compound or salt thereof obtained by means of the present inventioncan be used via the oral route as, for example, a tablet or pill,possibly sugar coated, a capsule, or a microcapsule, or can be used viaa parenteral route in the form of an injectable, for example, a sterilesolution or suspension with water or with a pharmaceutically acceptableliquid other than water. For example, production can be carried out bymixing a compound or salt thereof obtained by means of the presentinvention, in the unit dose form required for the elaboration of agenerally recognized formulation, with, for example, a physiologicallyacceptable carrier, flavorant, filler, vehicle, preservative,stabilizer, binder, and so forth.

The amount of effective component in these formulations is an amountthat provides an appropriate dose in the indicated range. Additives thatcan be incorporated into, for example, a tablet or capsule, can beexemplified by binders such as gelatin, corn starch, tragacanth, gumarabic, and so forth; fillers such as crystalline cellulose; swellingagents such as corn starch, gelatin, alginic acid, and so forth;lubricants such as magnesium stearate and so forth; sweeteners such assucrose, lactose, saccharin and so forth; and flavorants such aspeppermint, Japanese wintergreen (Gaultheria adenothrix) oil, andcherry. When the unit formulation takes the form of a capsule, startingmaterial of the aforementioned type may also include a liquid carriersuch as an oil or fat. Sterile compositions for injection can beformulated by the usual methods for producing a formulation, such asdissolving or suspending a natural vegetable oil or the like, such assesame oil or coconut oil, and the active ingredient in a vehicle suchas injection-grade water.

Examples of aqueous solutions for use as injection-grade water includephysiological saline, isotonic solutions containing glucose and/or otheradjuvants (such as D-sorbitol, D-mannitol, sodium chloride, and soforth), and suitable dissolution auxiliaries such as alcohols (e.g.,ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol, andso forth), and nonionic surfactants (e.g., Polysorbate 80™, HCO-50, andso forth) can be used in combination therewith. Sesame oil and soy oilare examples of dissolution auxiliaries, and, for example, benzylbenzoate, benzyl alcohol, and so forth can be used in combinationtherewith as dissolution auxiliaries. Buffers (e.g., phosphate buffers,sodium acetate buffers, and so forth), analgesics (e.g., benzalkoniumchloride, procaine chloride, and so forth), stabilizers (e.g., humanserum albumin, polyethylene glycol, and so forth), preservatives (e.g.,benzyl alcohol, phenol, and so forth), antioxidants, and the like mayalso be incorporated. The formulated injectable is usually filled into asuitable ampule.

A formulation obtained as described in the preceding is safe andexhibits low toxicity and can therefore be administered to, for example,mammals and warm-blooded animals (for example, human, rat, mouse, guineapig, rabbit). The administered dose of a compound or salt obtained bymeans of the present invention will vary as a function of, inter alia,the targeted disease, the recipient of the administration, and the routeof administration. As an example, when a compound or salt thereofobtained by means of the present invention is administered orally forthe purpose of treating hay fever, 0.1 mg to 1.0 g and preferably about1.0 mg to 50 mg of the compound or salt thereof is generallyadministered per day to an adult (60 kg).

EXAMPLES

The present invention is described in additional detail in the followingusing examples, but the present invention is not limited to theseexamples. The individual skilled in the art will be able to implementnot only the examples given in the following, but will also be able toadd various modifications; these modifications are also encompassed inthe claims provided herein.

Example 1

Measurement of the Transcriptional Regulatory Activity of the 5′ Regionof the Human FcεRI γ-chain Gene

nt-103 to -1 and nt-74 to -1 at the 5′ region of the human FcεRI γ-chaingene were each incorporated upstream from the luciferase gene in thepGL3Basic Vector (Promega), a plasmid that contains the fireflyluciferase gene as a reporter gene, to construct the respective reporterplasmids. 5 μg of the particular reporter plasmid and 0.1 μg pRL-CMVVector (Promega), a plasmid encoding the Renilla luciferase gene underCMV promoter control, as the control were transfected into each of fourγ-chain-expressing human cell lines (Jurkat, KU812, THP1, U937) byelectroporation (300 V, 950 μF).

After cultivation for 20 to 24 hours at 37° C./5% CO₂, the cells wererecovered and cell lysis and measurement of the luciferase activity werecarried out using a Dual Luciferase Assay Kit (Promega). At the time ofmeasurement, the value of firefly luciferase activity/Renilla luciferaseactivity was calculated for each sample and the plasmid transfectionefficiency and cell lysis efficiency were corrected.

The relative activity is shown in FIG. 1, where a value of 1 wasassigned to the luciferase activity for transfection with a reporterplasmid containing only the firefly luciferase gene. As shown in FIG. 1,in all of the γ-chain-expressing cell lines used, the -103 to -1 regioncaused a major enhancement in luciferase activity, while the -74 to -1region showed almost no enhancement effect of this nature. This exampledemonstrated that a transcription activating element with a commonfunction in the four cell lines is present in the -103 to -75 region.

Example 2

Identification of Factors Binding to nt-102 to -88

Gel shift assays were carried out using a nuclear extract prepared fromKU812 cells and the FITC-labeled double-stranded synthetic oligoDNAprobe 5′-ATGGGGGAAGGCGTG-3′ (corresponds to nt-102/-88 of the γ-chaingene).

Two unlabeled double-stranded synthetic oligoDNA's were used as thecompetitors: one having the same base sequence as the probe (comp) andone in which the three bases at nt-98 to -96 were changed (mut-comp).

30 μl of the aforementioned nuclear extract and 5 pmol of theaforementioned probe and 25 or 250 pmol of the competitor were mixed ina 10 mM HEPES buffer (pH 7.9) containing 400 ng poly(dl-dC), 1 mM MgCl₂,30 mM KCl, 1 mM DTT, and 5% glycerol and this was allowed to stand for20 minutes at room temperature. This was followed by submission to 4%polyacrylamide gel electrophoresis using 0.5×TBE buffer (45 mM Tris, 45mM boric acid, 1 mM EDTA). After phoresis for 2 to 3 hours at 120 V, theFITC fluorescence was detected using a FluorImager 595 (AmershamBioscience). The results are shown in FIG. 2(A).

As shown in FIG. 2(A), several bands were observed (lane 2) that wereshifted to positions of lower mobility than the band for the probe byitself. Among these, the band indicated by the arrow was extinguished ina competitor concentration-dependent manner when comp was added at 25and 250 pmol (lanes 3 and 4), while extinction of this band did notoccur in the case of the addition of mut-comp at 25 and 250 pmol (lanes5 and 6). This showed that this band was a band in which nuclear proteinwas bound with specific recognition of the sequence based on nt-98 to-96.

The same results were obtained for the use of nuclear extracts preparedfrom Jurkat, THP1, and U937.

In order to identify this nuclear protein, 2 μg antibody (Santa CruzBiotechnology, Inc.) against each of the transcription factors USF-1,USF-2, Sp1, and Ikaros was added. As shown in FIG. 2(B), the bandindicated by the arrow was extinguished only for the addition ofanti-Sp1 antibody. The same results were obtained for the use of nuclearextracts prepared from Jurkat, THP1, and U937. It was thus confirmedthat the transcription factor Sp1 binds to the sequence based on nt-98to -96.

Example 3

Identification of Factors Binding to nt-93 to -76

In order to identify factors binding to nt-93 to -76 of the human FcεRIγ-chain gene, a gel shift assay was carried out as in Example 2, but inthis case using the double-stranded synthetic oligoDNA probe5′GGCGTGGCAGGAAGAGGG-3′ as the probe and using, as the competitors, twounlabeled double-stranded synthetic oligo's, one having the same basesequence as the probe (comp) and one in which the three bases at nt-84to -82 were changed (mut-comp). The results are shown in FIG. 3(A).

As shown in FIG. 3(A), the two bands indicated by the arrows underwent acompetitor concentration-dependent extinction in the case of theaddition of 25 and 250 pmol comp (lanes 3 and 4), but were notextinguished in the case of the addition of 25 and 250 pmol mut-comp(lanes 5 and 6). This showed that these bands were bands in whichnuclear protein was bound with specific recognition of the sequencebased on nt-84 to -82. The same results were obtained for the use ofnuclear extracts prepared from Jurkat, THP1, and U937.

In order to identify this nuclear protein, 2 μg antibody (Santa CruzBiotechnology, Inc.) against each of the transcription factors PU.1,Fli1, Elf-1, GABP α, and GABP β was added. As shown in FIG. 3(B), of thetwo bands indicated by the arrows, the lower band was extinguished whenanti-GABP α antibody and anti-GABP β antibody were added, while theupper band was extinguished by the addition of anti-Elf-1 antibody. Thesame results were obtained for the use of nuclear extracts prepared fromJurkat, THP1, and U937. It was thus confirmed that GABP α/β heterodimerand Elf-1 bound to the sequence based on nt-84 to -82.

Example 4

Influence of Base Substitution by Site-directed Mutagenesis on γ-chainPromoter Activity

The nt-177 to -1 fragment of the human FcεRI γ-chain gene was insertedupstream from the firefly luciferase gene in the pGL3Basic Vector(Promega), and the γ-chain gene fragment in the resulting plasmid wassubjected to site-directed mutagenesis to construct four reporterplasmids. Thus, using a Quick Change Site-Directed Mutagenesis Kit(Stratagene), four reporter plasmids were obtained by replacing thefollowing three or four bases, respectively: nt-98 to -96 (mut1), -84 to-82 (mut2), -77/-75/-74 (mut3), and -65/-64/-62/-61 (mut4). Proceedingas in Example 1, expression testing was carried out by transfecting theobtained reporter plasmids into human cell lines (Jurkat, KU812, THP1,U937). The relative activity is shown in FIG. 4, where a value of 1 wasassigned to the luciferase activity for transfection with the reporterplasmid in which mutation had not been induced. As shown in FIG. 4, adecline in luciferase activity was observed for base substitution atnt-98 to -96, -84 to -82, and -65/-64/-62/-61, while the luciferaseactivity was unchanged for base substitution at -77/-75/-74.

The regions based on nt-98 to -96 and -84 to -82, which were shown inaccordance with the preceding Examples 2 and 3 to bind, respectively,Sp-1 and GABP α/β or Elf-1, were confirmed in accordance with thisexample to function as transcription activation elements. In addition,the region based on nt-65 to -61, which is downstream from thepreceding, was also shown to function as a transcription activationelement. The region based on nt-65 to -61 is homologous with the bindingmotif of the C/EBP transcription factor. Moreover, when these resultsare considered in combination with the results of Example 1, theconclusion is drawn that this element has almost no transcriptionactivating capacity by itself and functions cooperatively with the othertwo transcription activation elements.

Example 5

Influence of the Overexpression of Various Transcription Factors onγ-chain Promoter Activity

Expression tests were carried out as in Example 1 by transfecting HeLacells with 5 μg of a reporter plasmid prepared by inserting the nt-177to -1 region of the human FcεRI γ-chain gene upstream from the fireflyluciferase gene in the pGL3-Basic Vector (Promega) and with 3 μg of anexpression plasmid for GABP α, GASP β, Elf-1, Sp1, and/or C/EBP α.

The relative activity is shown in FIG. 5, where a value of 1 is assignedto the luciferase activity for transfection with only the reporterplasmid. The black bar in the graph in FIG. 5 shows the relativeactivity for transfection with the expression plasmid(s) for theparticular transcription factor(s), while the white bar shows therelative activity for transfection with the same amount of thecorresponding empty vector. As shown in FIG. 5, the luciferase activitywas increased several fold by the expression of GASP α/β only, or Elf-1only, or Sp1 only, or C/EBP α only, in comparison to transfection withthe empty vector, thus confirming that these transcription factors infact activate the γ-chain promoter. In addition, a synergisticactivation of the γ-chain promoter was shown for GABP α/β+Sp1+C/EBP αand for Elf−1+Sp1+C/EBP α.

1. An isolated nucleic acid molecule consisting of the nucleotidesequence of SEQ ID NO:1.
 2. A method of screening for a compound, orsalt thereof, that regulates the binding of Sp1 with an FcεRI γ-chaingene transcriptional regulatory region comprising, contacting thecompound with the isolated nucleic acid molecule of claim 1, andselecting a sample that binds to the isolated nucleic acid moleculewherein the presence of binding indicates that the compound regulatesthe binding of Sp1 with an FcεRI γ-chain gene transcriptional regulatoryregion.
 3. A screening kit comprising the isolated nucleic acid moleculeof claim
 1. 4. The nucleic acid molecule of claim 1, wherein the nucleicacid molecule regulates transcription of the human high affinity IgEreceptor (FcεRI) γ-chain gene.
 5. The method of claim 2, wherein themethod utilizes a gel shift assay or an antibody assay.